Roesley, S. N. A... Richardson, H. E. (2018). Phosphorylation of Drosophila Brahma on CDK-phosphorylation sites is important for cell cycle regulation and differentiation. Cell Cycle: 1-20. PubMed ID: 29963966Summary:
The SWI/SNF ATP-dependent chromatin-remodeling complex associates with the Retinoblastoma (pRb)/HDAC/E2F/DP complex to modulate cell cycle-dependent gene expression. The key catalytic component of the SWI/SNF complex in mammals is the ATPase subunit, Brahma (BRM) or BRG1. This study demonstrates the importance of CDK2-mediated phosphorylation of Brm in cell proliferation and differentiation in vivo using Drosophila. Expression of a CDK-site phospho-mimic mutant of Brm, brm-ASP (all the potential CDK sites are mutated from Ser/Thr to Asp), which acts genetically as a brm loss-of-function allele, dominantly accelerates progression into the S phase, and bypasses a Retinoblastoma-induced developmental G1 phase arrest in the wing epithelium. Conversely, expression of a CDK-site phospho-blocking mutation of Brm, brm-ALA, acts genetically as a brm gain-of-function mutation, and in a Brm complex compromised background reduces S phase cells. Expression of the brm phospho-mutants also affected differentiation and Decapentaplegic (BMP/TGFbeta) signaling in the wing epithelium. Altogether these results show that CDK-mediated phosphorylation of Brm is important in G1-S phase regulation and differentiation in vivo.

The endocycle provides a useful model for determining the minimum cell cycle inputs required to achieve a G/S oscillation and the once-per-cell-cycle replication of the genome. This study demonstrates that APC/C activity is required for endocycle progression. During the endocycle, mitotic activities are repressed. This is accomplished, at least in part, by preventing the accumulation of the mitotic activators Cyclin A, Cyclin B and Cdc25, which function to activate the mitotic kinase Cdk1. During the mitotic cycle, the mitotic cyclins are periodically targeted for regulated proteolysis by the E3-Ubiquintin ligase the APC/C. Yet the transcriptional downregulation of several APC/C targets at the mitotic/endocycle boundary, including the mitotic cyclins and String/Cdc25, suggested that the proteolytic activity of the APC/C might not be necessary during endocycles. However, this study found that compromising APC/C activity, after cells have entered the endocycle, results in the accumulation of Geminin and the mitotic cyclins, and in a block of DNA replication. Thus, the transcriptional downregulation of APC/C targets observed at the mitotic/endocycle transition is either downstream of APC/C activity and/or not sufficient to maintain low levels of these proteins. Taken together, these data suggest a model in which APC/C promotes the G/S oscillation of the endocycle by preventing the unscheduled accumulation of Geminin and the mitotic cyclins (Narbonne-Reveau, 2008).

During endocycles, APC/C activity prevents the inappropriate accumulation of Geminin, an inhibitor of the DNA replication-licensing factor Cdt1/Dup. When directly expressed in endocycling cells, Geminin efficiently inhibits DNA replication. These results strongly suggest that an essential function of the APC/C during the endocycle is to prevent the unregulated accumulation of Geminin. A similar role has been proposed for the APC/C during endoreplicative cycles of mouse trophoblasts (Gonzalez, 2006). However, the current data indicate that Geminin is not the only essential target of the APC/C during endocycles. A candidate for a second important target of the APC/C during endocycles is Cyclin A. Previous studies have shown that the overexpression of Cyclin A in the salivary gland, between the first and second endocycle, results in variable inhibitory effects on endoreplication. Although the majority of salivary gland cells that overexpress Cyclin A appear to be unaffected, a small percentage show a marked decrease in ploidy values. The reason for this variability is not clear. However, if the inhibitory influence of Cyclin A is mediated through binding and activation of Cdk1, this effect may be greatly amplified in the presence of high levels of String/Cdc25, which removes an inhibitory phosphate from Cdk1. Recent studies indicate that String/Cdc25, which contains both a consensus Ken box and D-box, is a target of the APC/C (Barbara Thomas, personal communication to Narbonne-Reveau, 2008). Therefore, an essential function of the APC/C during endocycles may involve restricting the activity of the mitotic kinase Cdk1, by preventing the accumulation of both Cyclin A and String/Cdc25. Finally, it is noted that the APC/C may have additional essential targets during the endocycle, which have yet to be identified (Narbonne-Reveau, 2008).

The periodic accumulation of the Orc1 protein during endocycles strongly suggests that the activity of the APC/CFzy/Cdh1 may not be continuous but cyclical. Previous work indicates that in Drosophila Cyclin E/Cdk2 inhibits the activity of APC/CFzy/Cdh1. These data are consistent with the observation that phosphorylation of Fzr/Cdh1 by Cdks inhibits the ability of Fzr/Cdh1 to bind and activate the APC/C in yeast, Xenopus and mammals. During the endocycle, the levels of Cyclin E oscillate. Taken together, these observations suggest a model in which APC/CFzy/Cdh1 is regulated by the periodicity of Cyclin E/Cdk2 activity, with high levels of Cyclin E resulting in the inhibition of APC/CFzy/Cdh1 activity and low levels of Cyclin E permitting full APC/CFzy/Cdh1 activity. The current data support this hypothesis. First, it was found that the periodicity of Orc1 levels during the endocycle requires a functional O-box, consistent with the cyclic destruction of Orc1 by APC/CFzy/Cdh1. Second, the levels of Orc1 are sensitive to Cyclin E. Specifically, overexpressing Cyclin E after cells have entered the endocycle results in the accumulation of APC/CFzy/Cdh1 targets, including Orc1, Cyclin A, Cyclin B and Geminin. Thus, the regulatory relationship observed between Cyclin E/Cdk2 and Fzr/Cdh1 that has been reported during mitotic cycles is conserved during endocycles. Finally, in endocycling cells the accumulation of Orc1 occurs during periods of high Cyclin E/Cdk2 activity, when APC/CFzy/Cdh1 dependent proteolysis would be predicted to be low. These data support the idea that the oscillations of Cyclin E/Cdk2 activity drive the periodicity of APC/CFzy/Cdh1 activity during the endocycle (Narbonne-Reveau, 2008).

Although a requirement for the oscillation of APC/CFzy/Cdh1 activity during the Drosophila endocycle has not been formally demonstrated, it is interesting to speculate on how the cyclic, rather than the continuous, activity of the APC/C might serve to facilitate endocycle progression. The data indicate that a period of high APC/CFzy/Cdh1 activity is required during the G phase of the endocycle in order to degrade the mitotic cyclins and Geminin, which can function to inhibit the formation of pre-RCs. However, a period of low APC/C activity may also be important. The continuous activation of APC/CCdh1 significantly slows DNA replication in mouse tissue culture cells. This inhibition may reflect the inability of a cell to accumulate adequate levels of proteins required for DNA replication, such as the APC/CCdh1 target and pre-replication complex component CDC6, in the presence of a constitutively active APC/CCdh1. In Drosophila, continuous APC/CFzy/Cdh1 activity might prevent the accumulation of two pre-RC components, CDC6 and Orc1. Intriguingly, APC/C activity also appears to oscillate during mammalian endocycles. In endocycling mouse trophoblasts, the levels of Cyclin A oscillate, consistent with the regulated destruction of the Cyclin A protein by the APC/C. Additionally, the inhibition of APC/C activity in endocycling trophoblasts results in the accumulation of the APC/C targets Cyclin A and Geminin. Taken together, these observations support a model in which the oscillation of APC/CFzy/Cdh1 activity, which is driven by the regulatory influences of Cdks, promotes efficient cell cycle progression during the endocycle (Narbonne-Reveau, 2008).

The data raises important questions. Why do levels of some APC/CFzy/Cdh1 targets, such as Cyclin A, Cyclin B and Geminin, remain below the level of detection while the levels of Orc1 protein oscillate? What might account for these different modes of regulation? Currently, there is no definitive explanation. However, at least three possibilities, which are not mutually exclusive, are envisaged, that may contribute to this differential behavior. First, it was found that relative to the Cyclin A and geminin, the levels of Orc1 transcript are only minimally downregulated upon entry into the endocycle. Transcriptional downregulation, or changes in transcript stability, may help contribute to the low levels of Geminin and Cyclin A proteins observed during the endocycle. Second, the translational efficiency of a subset of transcripts may be reduced upon entry into the endocycle. Finally, it is possible that the Orc1 protein is not as efficiently targeted by the APC/CFzy/Cdh1 as the mitotic cyclins or Geminin. Indeed the cis-acting sequences that target these proteins for destruction show considerable variability. Orc1 is targeted for APC/CFzy/Cdh1 destruction via a novel motif called the O-box (Araki, 2005). By contrast, Cyclin B and Geminin are targeted by a similar but unique sequence called the destruction-box (D-box), while Drosophila Cyclin A is targeted for destruction by a large complex N-terminal degradation sequence. There is precedence for post-translational regulation of APC/CFzy/Cdh1 targets, resulting in differential expression. In mammalian cells the pre-RC component CDC6, which is structurally related to Orc1, is protected from APC/CFzr/Cdh1 degradation by phosphorylation by Cyclin E/Cdk2. One or all of these potential mechanisms may contribute to the differential expression of various APC/CFzr/Cdh1 targets during the endocycle (Narbonne-Reveau, 2008).

Recent evidence from mice indicates that the depletion of the APC/C inhibitor Emi1/Rca1, results in both a strong decrease in E2F target mRNAs, such as geminin and Cyclin A, as well as APC/C activation. This study suggested that the regulation of APC/C activity, by the inhibitor Emi1/Rca1, drives a positive feedback circuit that controls both protein stability and mRNA expression. Thus, the observed decrease in the levels of at least some APC/C targets that occurs upon depletion of Emi1/Rca1, including Geminin and Cyclin A, are controlled at the levels of transcription and protein stability. Developmentally programmed endocycles may provide a natural example where cell cycle progression occurs in the context of increased APC/CFzr/Cdh1 activity. Thus, a similar positive-feedback circuit may be operating during Drosophila endocycles to downregulate the transcription of E2F target genes. Determining the precise regulatory relationships between the upregulation of APC/CFzr/Cdh1 activity and the transcriptional downregulation of genes such as Cyclin A and geminin, during the Drosophila endocycle represents an exciting area for future research (Narbonne-Reveau, 2008).

The requirement for APC/C activity to promote endocycle progression may help answer several longstanding questions concerning the regulation of the Drosophila endocycle. For example, why does the continuous expression of Cyclin E inhibit cell cycle progression during the endocycle but not the mitotic cycle? Several models have been proposed to explain this difference. First, the breakdown of the nuclear envelope that occurs during the mitotic cycle, but not the endocycle, may allow for a transient decrease in local Cyclin E/Cdk2 activity, thus allowing for the relicensing of DNA replication origins. Alternatively, there may be differences in the machinery required to produce a functional pre-RC in mitotic versus endocycling cells. The current results suggest an alternative model for why endocycles are unusually sensitive to continuous Cyclin E expression. This model is based on the demonstration that endocycle progression requires APC/C activity. Both Fzy/Cdc20 and Fzr/Cdh1 function as activators of the APC/C. However, the regulation of these APC/C activators is very distinct. During the mitotic cycle, the binding of Fzy/Cdc20 to the APC/C is dependent on the phosphorylation of several APC/C subunits by the mitotic kinase Cdk1. By contrast, a Cdk-dependent inhibitory phosphorylation on Fzr/Cdh1 relegates APC/CFzr/Cdh1 activity to late M phase and G1. Because of its requirement for Cdk1 activity, APC/CFzy/Cdc20 is unlikely to be active during most endocycles. Indeed, Drosophila endocycles proceed normally in fzy mutants. Thus, the only available activator of the APC/C during the endocycle is Fzr/Cdh1. As previously discussed, Fzr/Cdh1 is inhibited by Cyclin E/Cdk2 activity. Therefore, it is proposed that during the endocycle, continuous Cyclin E/Cdk2 activity results in the permanent inhibition of the only available activator of the APC/C, Fzr/Cdh1. This leads to the accumulation of Geminin, Cyclin A and other potential targets, which act to block cell cycle progression. Thus, the ability of continuous Cyclin E to inhibit DNA replication during the endocycle may reflect differences in the available activators of the APC/C present in mitotic versus endocycling cells (Narbonne-Reveau, 2008).

Integrated stability and activity control of the Drosophila Rbf1 retinoblastoma protein

The retinoblastoma (RB) family transcriptional corepressors regulate diverse cellular events including cell cycle, senescence, and differentiation. The activity and stability of these proteins are mediated by post-translational modifications; however, a general understanding of how distinct modifications coordinately impact both of these properties is lacking. It has been shown that protein turnover and activity are tightly linked through an evolutionarily conserved C-terminal instability element (IE) in the Drosophila RB-related protein Rbf1; surprisingly, mutant proteins with enhanced stability were less, not more active. To better understand how activity and turnover are controlled in this model RB protein, this study assessed the impact of Cyclin-Cdk kinase regulation on Rbf1. An evolutionarily conserved N-terminal threonine residue is required for Cyclin-Cdk response, and a dominant impact on turnover and activity was demonstrated; however, specific residues in the C-terminal IE differentially impacted Rbf1 activity and turnover, indicating an additional level of regulation. Strikingly, specific IE mutations that impaired turnover but not activity induced dramatic developmental phenotypes in the Drosophila eye. Mutation of the highly conserved Lys-774 residue induced hypermorphic phenotypes that mimicked the loss of phosphorylation control; mutation of the corresponding codon of the human RBL2 gene has been reported in lung tumors. These data support a model in which closely intermingled residues within the conserved IE govern protein turnover, presumably through interactions with E3 ligases, and protein activity via contacts with E2F transcription partners. Such functional relationships are likely to similarly impact mammalian RB family proteins, with important implications for development and disease (Zhang, 2014).

The mob as tumor suppressor gene is essential for early development and regulates tissue growth in Drosophila

Studies in Drosophila have defined a new growth inhibitory pathway mediated by Fat (Ft), Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav)/Shar-pei, Warts (Wts)/Large tumor suppressor (Lats), and Mob as tumor suppressor (Mats), which are all evolutionarily conserved in vertebrate animals. The Mob family protein Mats functions as a coactivator of Wts kinase. This study shows that mats is essential for early development and is required for proper chromosomal segregation in developing embryos. Mats is expressed at low levels ubiquitously, which is consistent with the role of Mats as a general growth regulator. Like mammalian Mats, Drosophila Mats colocalizes with Wts/Lats kinase and cyclin E proteins at the centrosome. This raises the possibility that Mats may function together with Wts/Lats to regulate cyclin E activity in the centrosome for mitotic control. While Hpo/Wts signaling has been implicated in the control of cyclin E and diap1 expression, this study found that it also modulates the expression of cyclin A and cyclin B. Although mats depletion leads to aberrant mitoses, this does not seem to be due to compromised mitotic spindle checkpoint function (Shimizu, 2008).

Mats is essential for normal development; mats mutants stop their growth at the second instar larval stage and eventually die. In fact, this growth retardation phenotype facilitated identification of matsroo and matse235 mutant larvae for DNA sequence analysis. Using matse235 allele and the P-element-induced allele matsPB, it has been shown that mats homozygotes and hemizygotes grow slowly and their imaginal discs are much smaller than that of wild-type larvae at the same age. mats mutant cells in mosaic tissues acquire growth advantage likely through comparison and competition with neighboring wild-type cells. In contrast, the absence of wild-type cells in homozygous mats mutant animals renders no competitive growth advantage to mutant cells. The mechanism by which mats mutants acquire growth advantage in the context of mosaic tissue still needs to be investigated. mats mutant embryos missing both maternal and zygotic mats functions failed to hatch, indicating that mats is essential for embryonic development. By analyzing mitotic cells, it was found that maternally mats-depleted embryos show aberrant DNA segregation such that uneven amounts of DNA are segregated toward opposing centrosomes. However, this does not appear to be due to the compromised function of mitotic spindle checkpoint, since mats mutant tissue still accumulate M-phase cells in response to inhibition of mitotic spindle formation by colcemid treatment. Thus, mats is not required for mitotic spindle checkpoint, unlike mps1 (Shimizu, 2008).

Cyclin E is a critical cell cycle regulator. Through a Cdk2-dependent mechanism, cyclin E-Cdk2 plays a critical role in accelerating G1-S transition in the cell cycle. As a general rule, cyclin E is tightly regulated during the cell cycle by Cdk2 and GSK-mediated phosphorylation and subsequent degradation. A nondegradable cyclin E mutant can cause extra rounds of DNA synthesis and polyploidy, and overexpression of cyclin E is frequently detected in tumor cells exhibiting polyploidy. Intriguingly, cyclin E is a centrosomal protein that functions to promote S-phase entry and DNA synthesis in a Cdk2-independent manner (Matsumoto, 2004). Loss of cyclin E expression in the centrosome inhibits DNA synthesis, whereas ectopic expression of cyclin E in the centrosome accelerates S-phase entry. Thus, the centrosome is an important subcellular organelle for cyclin E to regulate cell proliferation, and the level and activity of cyclin E in centrosomes must be tightly controlled. The fact that Mats and Wts colocalize with cyclin E at the centrosome raises the possibility that Mats may function together with Wts kinase to regulate cyclin E function in the centrosome for mitotic control. In support of this hypothesis, loss-of-function mutations in mats increase the levels of cyclin E protein and both gain- and loss-of-function mutant alleles of cyclin E modulate the eye phenotypes caused by Wts overexpression. Although Mats/Wts-mediated inhibition of cyclin E could occur through Yki to regulate cyclin E transcription, a direct control of cyclin E at the protein level would allow a rapid response to an upstream signal (Shimizu, 2008).

The fact that both Mats and Wts show a intracellular localization pattern very similar to that of their respective yeast relatives Mob1 and Dbf2 suggests that their function is conserved. This conservation may extend to mammals; human LATS1, LATS2, and MOB1A (MATS2) also localize at the centrosome. In addition, localization at the bud neck/midbody appears to be conserved in humans. Interestingly, such centrosomal localization of Mats and Wts does not seem to rely on Wts kinase activity as kinase-inactive Wts and Mats can be still localized at the centrosome. To examine whether endogenous Mats protein localizes at the centrosome, embryo immunostaining was done with Mats antibodies. As in larval tissues, expression of Mats protein in developing embryos does not exhibit any obvious pattern and Mats expression level is low and ubiquitous. Although centrosomal localization of endogenous Mats protein has not been shown, likely due to some technical problems, Mats (CG13852/Mob4) has been recently reported to be a centrosomal protein (Shimizu, 2008 and references therein).

Both loss- and gain-of-function analysis supports a model in which cyclin E and diap1 are critical downstream targets of Hpo/Wts signaling. Evidence in this report suggests that Hpo/Wts signaling may also target cyclin A and cyclin B. Consistent with this notion, elevated levels of cyclin B were found in ex mutant cells. In addition, wts has been shown to be required for a negative control of cyclin A but not cyclin B expression. In humans, LATS1 was shown to be a negative regulator of Cdc2/cyclin A and to function at the G2/M-phase transition, while LATS2 affects cyclin E/Cdk2 activity and regulates G1/S phase passage. Thus, the ability of Hpo/Wts signaling to target cyclin genes important for cell cycle progression appears to be evolutionarily conserved (Shimizu, 2008).

Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).

Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).

How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).

More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).

Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).

In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).

How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21Cip1 and p27Kip1 (Buttitta, 2007).

There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19Ink4d and p27Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19Ink4d, p27Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).

The endocycle is a developmentally programmed variant cell cycle in which cells undergo repeated rounds of DNA replication with no intervening mitosis. In Drosophila, the endocycle is driven by the oscillations of Cyclin E/Cdk2 activity. How the periodicity of Cyclin E/Cdk2 activity is achieved during endocycles is poorly understood. This study has demonstrated that the p21cip1/p27kip1/p57kip2-like cyclin-dependent kinase inhibitor (CKI), Dacapo (Dap), promotes replication licensing during Drosophila endocycles by reinforcing low Cdk activity during the endocycle Gap-phase. In dap mutants, cells in the endocycle have reduced levels of the licensing factor Double Parked/Cdt1 (Dup/Cdt1), as well as decreased levels of chromatin-bound minichromosome maintenance (MCM2-7) complex. Moreover, mutations in dup/cdt1 dominantly enhance the dap phenotype in several polyploid cell types. Consistent with a reduced ability to complete genomic replication, dap mutants accumulate increased levels of DNA damage during the endocycle S-phase. Finally, genetic interaction studies suggest that dap functions to promote replication licensing in a subset of Drosophila mitotic cycles (Hong, 2007).

Levels of the origin-binding protein Double parked and its inhibitor Geminin increase in response to replication stress

The regulation of a pre-replicative complex (pre-RC) at origins ensures that the genome is replicated only once per cell cycle. Cdt1 is an essential component of the pre-RC that is rapidly degraded at G1-S and also inhibited by Geminin (Gem) protein to prevent re-replication. Destruction of the Drosophila homolog of Cdt1, Double-parked (Dup), at G1-S is dependent upon cyclin-E/CDK2 and important to prevent re-replication and cell death. Dup is phosphorylated by cyclin-E/Cdk2, but this direct phosphorylation is not sufficient to explain the rapid destruction of Dup at G1-S. Evidence is presented that it is DNA replication itself that triggers rapid Dup destruction. A range of defects in DNA replication stabilize Dup protein and this stabilization is not dependent on ATM/ATR checkpoint kinases. This response to replication stress is cell-type specific, with neuroblast stem cells of the larval brain having the largest increase in Dup protein. Defects at different steps in replication also increased Dup protein during an S-phase-like amplification cell cycle in the ovary, suggesting that Dup stabilization is sensitive to DNA replication and not an indirect consequence of a cell-cycle arrest. Finally, it was found that cells with high levels of Dup also have elevated levels of Gem protein. It is proposed that, in cycling cells, Dup destruction is coupled to DNA replication and that increased levels of Gem balance elevated Dup levels to prevent pre-RC reformation when Dup degradation fails (May, 2005).

double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2

It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. The developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms, has been studied. Phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, these results indicate that stringent regulation of Dup protein is critical to protect genome integrity (Thomer, 2004).

To determine whether oscillation of Dup protein levels during cell cycles is due to Dup protein degradation at G1/S, Dup expression within the synchronized cell cycles of the larval eye primordium was examined. Late in third instar, a wave of differentiation sweeps across the eye imaginal disc, which is visible as a morphogenetic furrow (MF). Cells are synchronized in G1 upon entering the furrow. Specific cells posterior to the furrow then enter a synchronous S phase, which is visible as a stripe of BrdU labeling. Labeling with affinity-purified rabbit polyclonal Dup antibody indicates that the protein is abundant in nuclei of late G1 cells, but is undetectable in S phase cells incorporating BrdU. Labeling with a guinea pig anti-Dup antibody gave identical results suggesting that immunolabeling reflects Dup protein in vivo. Double labeling for Dup and cyclin E indicates that both are abundant in nuclei of cells in late G1, but then Dup rapidly declines while cyclin E persists into S phase. Labeling for the G2 and M phase marker cyclin B also indicates that Dup levels decline significantly before cells enter G2. Similar results were obtained for the non-synchronized cell cycles in the eye and other imaginal discs. This rapid decline in protein is primarily due to post-transcriptional regulation because in situ hybridization indicates that dup mRNA persists after G1. Moreover, expression of a dup transgene from the strong hsp70 promoter does not result in detectable Dup protein during S phase. The data suggest that, similar to Cdt1 in humans and other organisms, Dup protein is abundant in G1 when origins are licensed, but is then rapidly degraded when cyclin E appears at G1/S (Thomer, 2004).

Beginning in late mitosis, origins of replication are prepared for replication by binding of a pre-replicative complex (pre-RC), which is subsequently activated to initiate replication at the onset of S phase. The building of the pre-RC onto origins in late mitosis/early G1 is a stepwise process. The origin recognition complex (ORC) serves as a scaffold for subsequent association of Cdc6 and Cdt1, both of which are required to load the Minichromosome Maintenance (MCM) complex, the replicative helicase. Once MCMs are loaded, the origin is considered to be licensed for subsequent replication. Cdc7 kinase, with its activating subunit Dbf4, and CDK2 kinase, activated by cyclin E or cyclin A, are then required for initiation of replication. Initiation is associated with departure of Cdc6, Cdt1, MCMs, and, in multicellular eukaryotes, certain ORC subunits from the origin. Continued CDK activity in S, G2, and early M phases inhibits reassembly of the pre-RC to block origin refiring. Unique to multicellular eukaryotes is another inhibitor of pre-RC assembly, Geminin, which binds Cdt1 and renders it incapable of loading the MCM complex. It is only after Geminin and cyclins are degraded at the subsequent metaphase that the pre-RC can reform, thereby restricting origin licensing, and DNA replication, to once per segregation of chromosomes (Thomer, 2004 and references therein).

Although phosphorylation of pre-RC subunits appears to be important for initiation and to block pre-RC re-assembly, the biochemical mechanisms are not fully understood. In the yeasts Saccharomyces cerevisiae and S. pombe, CDKs block re-replication by phosphorylating several pre-RC targets including CDC6 and subunits of the ORC and MCM complex. All three of these blocks must be abrogated before even partial re-replication is permitted in S. cerevisiae cells in G2, suggesting that multiple reinforcing mechanisms have evolved to protect the integrity of the genome. In S. pombe, however, over-expression of Cdc18 (the Cdc6 homolog) alone, but not other pre-RC subunits, is sufficient to induce re-replication. Thus, whether mis-regulation of a single protein can induce re-replication may differ among organisms. In higher eukaryotes, it also appears that CDKs block re-replication by targeting multiple pre-RC subunits to protect genome integrity (Thomer, 2004 and references therein).

Despite the prevailing concept of redundant controls, recent evidence suggests that regulation of Cdt1 is especially important to inhibit re-replication. In a number of systems, over-expression of Cdt1, or inactivation of its inhibitor Geminin, causes partial, but not full, re-replication of the genome. In all organisms examined, except S. cerevisiae, the majority of Cdt1 protein is rapidly degraded at the G1/S transition. Evidence from several organisms suggests that Cdt1 is targeted for degradation at the proteasome by two ubiquitin ligase complexes, an SCF (Skp1, Cul1, F box) ubiquitin ligase that contains the specificity subunit Skp2, and an SCF-like ubiquitin ligase that is based on Cul4 (see Drosophila Cul4). This degradation is probably important because over-expression of Cdt1 in p53 mutant human cells in culture can lead to partial re-replication, and contributes to oncogenic transformation of mouse erythroid cells. In Caenorhabditis elegans, RNAi of Cul4 leads to stabilization of Cdt1 protein and polyploidization. It is unclear, however, whether Cul4 controls degradation of other proteins important for re-replication control. Therefore, two important remaining questions are whether increased Cdt1 protein is sufficient to induce genome reduplication in normal cells during development, and what coordinates the rapid degradation of Cdt1 with the initiation of DNA replication at the G1/S transition (Thomer, 2004 and references therein).

The Drosophila ortholog of Cdt1, the double-parked (dup) gene, was initially identified as recessive embryonic lethal or female-sterile mutants that have defects in genomic replication or developmental amplification of eggshell (chorion) protein genes in the ovary. Evidence is provided that degradation of Dup is controlled in part by cyclin E/CDK2 phosphorylation, and additional mechanisms also ensure Dup degradation. Control of Dup protein abundance is critical because increased expression of Dup in diploid cells is sufficient to induce polyploidization and cell death in developing tissues. Interestingly, over-expression of wild-type and mutant Dup derivatives have different effects on genomic replication than on amplification from chorion origins. These last results provide insight into how phosphorylation regulates Dup during these developmentally distinct replication programs, and suggest that Dup participates in replication fork elongation during amplification (Thomer, 2004).

Therefore, phosphorylation and stability of Dup depends on cyclin E/CDK2 activity. It is likely that part of this regulation is direct because Dup associates with CDK2 protein and activity in embryos. The results of the mutagenesis show that the N terminus of Dup is necessary and sufficient for degradation at G1/S. Mutation of the CDK sites in the N terminus, however, only partially stabilize the protein, suggesting the existence of other CDK2-dependent mechanisms for degradation. It is crucial to tightly regulate the abundance of Dup protein because its over-expression is sufficient to induce a full genome reduplication and cell death in the ovary and imaginal discs. The different effects on amplification and genomic replication suggest that phosphorylation of the N terminus of Dup protein may be required for replication fork elongation during amplification and provides insight into the mechanism of this developmentally specific replication program (Thomer, 2004).

The results suggest that cyclin E/CDK2 phosphorylates the Dup N terminus contributing to its instability at G1/S. Dup was degraded during periodic endocycle S phases that are solely regulated by oscillating cyclin E/CDK2, further supporting a link between this kinase and Dup degradation. Although the N terminus was necessary and sufficient for degradation, mutation of the ten N-terminal CDK sites within Dup 10(A) only partially stabilized the protein. This suggests that there are other cyclin E/CDK2-dependent mechanisms that trigger Dup degradation independent of these ten sites during S phase. It has been noted that the C terminus of Dup contains a PEST sequence, and there are several serines and threonines in the C terminus that are potential targets of phosphorylation. Although the requirement for these sites has not been directly tested, the stability of C-Dup indicates that they are not sufficient for degradation at G1/S. To explain these results, a bi-phasic degradation model is suggested where cyclin E/CDK2 phosphorylation promotes Dup degradation in late G1, whereas other fail-safe mechanisms become operative only during S phase. This would explain why inhibiting CDK2 and S phase entry with GMRp21 completely blocked Dup degradation (Thomer, 2004).

A number of recent publications describe results for Cdt1 in human cells that are similar to those in flies. These results suggest that cyclin A/CDK2 phophorylates the human Cdt1 N terminus, which enhances its binding to the Skp2 subunit of the SCF ubiquitin ligase. Like Dup, non-phosphorylatable Cdt1 mutants are only partially stabilized, but simultaneously inhibiting CDK2 and S phase entry with p21 completely blocks degradation. Previous evidence in C. elegans, human and Drosophila cells have suggested that destruction of Cdt1 may be mediated by two ubiquitin ligases, an SCF complex containing Skp2, and an SCF-like complex based on Cul4. For many substrates of the SCF, prior phosphorylation is required for their subsequent recognition and ubiquitinylation, including substrates phosphorylated by CDK2 at G1/S. It is not known whether prior phosphorylation is required for substrate recognition by Cul4-based ubiquitin ligases. It is tempting to speculate, therefore, that the bi-phasic degradation of Cdt1 that may reflect its modification by two distinct ubiquitin ligases: a phosphorylation-dependent ubiquitinylation by the SCF complex, and a phosphorylation-independent ubiquitinylation by a Cul4-based complex. Clearly, more experiments are needed to sort out the complexity of this regulation. Nonetheless, the similar results from flies and humans suggest that tight regulation of Cdt1 abundance is a generally conserved and important mechanism to protect genome integrity in eukaryotes (Thomer, 2004).

CDK activity and Geminin play central roles in the block to re-replication. The results reported in this study show that Dup over-expression is sufficient to induce a full genome reduplication in normal cells in developing tissues, transforming diploid into polyploid cells. This phenotype is more profound than that of Geminin mutants, suggesting that degradation of Dup protein is of highest priority to protect genome integrity. An important caveat is that in these experiments Dup is over-expressed and therefore not equivalent to an absence of degradation. It was found, however, that even small, undetectable increases in Dup protein can have profound consequences. Moreover, after multiple heat pulses, Dup protein was undetectable during S phase, yet it induced extensive re-replication in most cells. The prolonged genomic replication in the synchronized cells of the eye disc suggests that this small increase in Dup protein may permit origins to be relicensed and reinitiate within a single S phase. While the precise molecular mechanism for how increased Dup promotes re-replication remains undefined, the results indicate that even a small increase in Dup protein is sufficient to compromise genome integrity (Thomer, 2004).

The other phenotype associated with over-expression of Dup is cell death. Dup
10(A), created by mutating the serines and threonines at putative phosphorylation sites to alanine, causes more cell death than wild-type Dup, suggesting that phosphorylation of the Dup N terminus influences this phenotype. In human cells re-replication due to over-expression of Cdt1 is more easily detected when p53 is mutant, probably because they escape apoptosis triggered by re-replication. Therefore the model is favored that Dup over-expression induces re-replication, which in turn can lead to the activation of checkpoints and apoptosis (Thomer, 2004).

In recent years, the analysis of replication from the defined chorion amplification origins has been a prominent genetic and molecular model system for the regulation of DNA replication in metazoa. Chorion origins require pre-RC proteins, cyclin E/CDK2 and Dbf4/Cdc7 kinases, indicating that their regulation resembles that of genomic origins. They clearly differ, however, in that they re-replicate at a time when no other origins are firing, and understanding this exception should provide insight into the rules of regulation of all origins. Surprisely, the carboxyl-terminal half of Dup, although having no effect on genomic replication, is a hyperactive protein that causes over-amplification from chorion origins. The Dup 10(A) mutant gave the opposite result; it was dominant negative and strongly inhibited amplification. It is proposed that during amplification phosphorylation of the Dup N terminus abrogates its inhibition of the activity of the C terminus, explaining why deleting the N terminus results in a hyperactive protein, whereas blocking its phosphorylation results in an inactive protein. An important functional role for the C terminus is consistent with its binding to MCM proteins, and the fact that among Cdt1 family members the C-terminal half is much more highly conserved than the N-terminal half of the protein. Most Cdt1 proteins have known or potential CDK phosphorylation sites in their N terminus, despite its poor conservation, supporting the notion that its conserved function is to mediate regulation by CDKs (Thomer, 2004).

The different effects on amplification versus genomic replication suggest a distinction in the regulation or function of Dup in these two processes. It has been proposed that Dup participates in fork elongation during amplification, based on immunolabeling at chorion foci. This study shows that in other cell cycles Dup is rapidly degraded at the onset of S phase and not present during fork elongation, similar to results from human and other cells. Moreover, S. cerevisiae cells experimentally depleted of Cdt1 within S phase are able to complete genomic replication, inconsistent with a role in elongation. Expression of Dup 10(A), however, inhibits BrdU incorporation within 1 hour in all stages of amplification, including late stages when only elongation of forks is occurring. This rapid and complete inhibition of BrdU incorporation by Dup 10(A) cannot be an indirect effect of origin inhibition, and supports the proposed role for Dup at the replication fork. Furthermore, this suggests that phosphorylation is important for the function of Dup in elongation during amplification. The distinct activities of C-Dup and Dup 10(A) in genomic replication versus amplification provide a molecular handle on the mechanism by which these two developmental replication programs differ, possibly resulting from the activity of Dup at the fork. The function of Dup at the fork may be related to its known ability to load the MCM complex helicase onto chromatin. It also raises the possibility that Cdt1 family members may act at the fork under other special circumstances (Thomer, 2004).

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Animal oocytes undergo a highly conserved developmental arrest in prophase
of meiosis I. Often this marks a period of rapid growth for the oocyte and is
necessary to coordinate meiotic progression with the developmental events of
oogenesis. In Drosophila, the oocyte develops within a 16-cell
germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of
meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become
polyploid in preparation for their role as nurse cells. How germline cysts
establish and maintain these two independent cell cycles is unknown. This study
demonstrates a role for the
p21CIP/p27Kip1/p57Kip2-like cyclin-dependent
kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle
in Drosophila oocytes. The data indicate that it is through the
differential regulation of the cki Dacapo that two modes of cell-cycle
regulation are independently maintained within the common cytoplasm of ovarian
cysts (Hong, 2003).

In females homozygous for the hypomorphic mutation
cycE01672, a fraction of egg chambers contain two cells
that have oocyte-like nuclear features, such as low ploidy values, an endobody
and a small DNA mass in a very large nucleus.
Egg chambers that contain two oocyte nuclei have only 14 polyploid nurse
cells, indicating that a cell that was destined to develop as a nurse cell has
been partially transformed towards the oocyte fate. The extra oocyte nucleus
(which can be distinguished from the true oocyte by its presence in a small
cell that lacks signs of cytoplasmic oocyte differentiation), almost
invariably is the other four-ring canal cell in the cyst.
Interestingly, these transformed nuclei accumulate persistently high levels of
Dap protein in a manner similar to the true oocyte. Cells with
persistently high levels of Dap have low ploidy values, indicating they have
either not entered the endocycle or have prematurely exited the cycle. By
contrast, in wild-type egg chambers the other four-ring canal cell develops as
a highly polyploid posterior nurse cell in which Dap levels oscillate. Thus,
mutations in dap result in germline cysts in which all 16 cells enter
the endocycle and develop as nurse cells, while a mutation in cycE
has the opposite effect, resulting in two or more cells that have persistently
high levels of Dap that cannot enter and/or maintain the endocycle (Hong, 2003).

To examine the relationship between cycE and dap in the
regulation of the cell-cycle program of ovarian cysts, whether
mutations in dap could dominantly modify the
cycE01672 two oocyte phenotype was examined. Reducing the
dose of the dap gene by half, results in ~2.5 fold suppression
of the cycE01672 two oocyte phenotype. In wild-type egg
chambers the four posterior nurse cells, which are connected to the oocyte via
ring canals, have the highest ploidy values in the cyst. In
cycE01672 females 37±7% of egg
chambers contain a cell adjacent to the true oocyte with inappropriately low
ploidy values. When a single copy of the null allele dap4 was placed in the cycE01672 background, fewer than 14±7%
of egg chambers had a posterior nurse cell with a reduced DNA content. These data indicate that whether a cyst cell enters and/or maintains the endocycle is at least partially determined by the balance of CycE and Dap. In addition, they strongly suggest that, as is observed during embryogenesis, the primary target of Dap in the ovary is the CycE/Cdk2 complex (Hong, 2003).

These observations suggest a model for how the meiotic cycle and the
endocycle are independently maintained within Drosophila ovarian
cysts. It is proposed that the presence of high levels of the cki Dap in the oocyte (throughout the time the nurse cells are in the endocycle) persistently inhibits cyclinE-Cdk2 kinase activity and prevents inappropriate DNA replication during meiosis. Without the inhibition of cyclinE-Cdk2 kinase activity provided by high levels of Dap, the majority of dap mutant oocytes abandon the meiotic cycle
and enter the endocycle with the nurse cells. Importantly, these data indicate
that, as has recently been observed in mice, oocytes in prophase of meiosis I
are competent to replicate their DNA. In the mouse oocyte, the inhibition of DNA replication during prophase of meiosis I may be accomplished through the downregulation of the G1 cyclins and Cdk2. In the Drosophila oocyte, the inhibition of cyclinE-Cdk2 activity by Dap achieves the same aim. In contrast to the oocyte, the nurse cells require a period when Dap levels are low to allow cyclinE-Cdk2 kinase activity to rise high enough to trigger each endocycle S phase. These low points occur during the oscillations of the Dap protein. The data indicate that it is through the differential regulation of Dap that two apparently incompatible cell cycles are stably maintained within the common cytoplasm of the ovarian cyst (Hong, 2003).

The regulatory relationship between cyclinE-Cdk2 activity and the Dap
ortholog p27 suggest a feedback loop that may account for the long-term
stabilization of Dap in post germarial oocytes. In mammalian cells,
phosphorylation by cyclinE-Cdk2 targets the p27 protein for destruction by the
proteasome. Similarly, the Dap protein contains a CDK phosphorylation
consensus site (Ser205) and can be phosphorylated by mammalian
cyclinE-Cdk2 in vitro. It is proposed that in early stage 1 egg chambers, the balance of cyclinE-Cdk2 activity and Dap protein is slightly different in the 15-nurse cells versus the single oocyte. In the oocyte, the balance is tipped towards the inhibitor Dap, resulting in diminished cyclinE-Cdk2 activity. Lower cyclinE-Cdk2
activity leads to a reduced rate of Dap phosphorylation and proteolysis,
thereby increasing the concentration of the Dap protein. The stabilization of
the Dap protein ultimately results in the permanent inhibition of cyclinE-Cdk2
activity in the oocyte. In contrast to the oocyte, in stage 1 nurse cells
cyclinE-Cdk2 kinase activity reaches high enough levels to trigger the
phosphorylation and subsequent destruction of the Dap protein, thus allowing
endocycle progression. The above model predicts that additional proteins that
are targeted for destruction by cyclinE-Cdk2 phosphorylation should be
stabilized in the oocyte but not in the nurse cells. Like p27, the proteolytic
destruction of CycE itself is dependent on phosphorylation by the cyclinE-Cdk2
complex. As predicted by the model, CycE is stabilized in the oocyte and accumulates to high levels as oogenesis progresses. This model allows the amplification of a slight difference in the balance of cyclinE-Cdk2 activity and Dap early in oogenesis, resulting in the two cell types of the germline cyst permanently adopting
dramatically different cell cycles (Hong, 2003).

A screen for modifiers of Cyclin E function in Drosophila melanogaster
identifies Cdk2 mutations, revealing the insignificance of putative
phosphorylation sites in Cdk2

To identify genes interacting with cyclin E, a screen was carried out for mutations that act as dominant modifiers of an eye phenotype caused by a Sevenless-CycE transgene that directs ectopic Cyclin E expression in postmitotic cells of eye imaginal disc and causes a rough eye phenotype in adult flies. The majority of the EMS-induced mutations that were identified fall into four complementation groups corresponding to the genes split ends (spen), dacapo, dE2F1, and Cdk2 (Cdc2c). The Cdk2 mutations in combination with mutant Cdk2 transgenes have allowed the regulatory significance of potential phosphorylation sites in Cdk2 (Thr 18 and Tyr 19) to be addressed. The corresponding sites in the closely related Cdk1 (Thr 14 and Tyr 15) are of crucial importance for regulation of the G2/M transition by myt1 and wee1 kinases and cdc25 phosphatases. In contrast, the results presented here demonstrate that the equivalent sites in Cdk2 play no essential role. The demonstration that phosphorylation of Cdk2 on Thr 18 and Tyr 19 has no essential role during normal development does not exclude its involvement in subtle or stress regulation. Moreover, vertebrate cells, in which Cdk2 phosphorylation on Thr 18 and Tyr 19 has been demonstrated to occur, express A-type cdc25 phosphatases that have been implicated in Cdk2 dephosphorylation and that do not appear to exist in Drosophila (Lane, 2000).

To characterize the effects of Cdk2 mutations on development, embryogenesis was examined. However, a requirement for zygotic Cdk2 expression during this developmental phase could not be identified. Embryos hemizygous or transheterozygous for Cdk2 allele combinations appear to have wild-type morphology. They incorporated BrdU with normal efficiency and in normal patterns throughout embryogenesis. Hatching of larvae has been observed to occur at the same rate as in control embryos (Lane, 2000).

For the characterization of larval development, progeny were analyzed from parents with Cdk2 alleles over a balancer chromosome carrying Tb. Scoring for the Tb phenotype, which can readily be distinguished from wild type after development beyond the second larval instar, suggests that Cdk2 mutants do not reach this stage. However, experiments involving the expression of Hs-Cdk2 in Cdk2 mutant larvae indicates that these mutants survive for longer time periods than what is normally required to reach second instar. As indicated above, periodic Hs-Cdk2 expression by heat shocks allows Cdk2 mutants to develop into morphologically normal and fertile adults. Some Cdk2 mutants are still observed to develop into adults even if the onset of the periodic Hs-Cdk2 expression is delayed until 116 hr after egg deposition. Cdk2 mutant larvae, therefore, fail to grow but some survive for several days and can be rescued by providing Cdk2 again. The dependence of larval growth on zygotic Cdk2 expression is confirmed by comparing the size of Cdk2+ and Cdk2 mutant larvae derived from parents with Cdk2 alleles over a balancer chromosome marked by an Act-GFP transgene. GFP-negative Cdk2 mutant larvae are observed in decreasing numbers for at least 4 days after egg deposition, but their size fails to increase significantly after 1.5 days (Lane, 2000).

The normal initial development that is observed in the absence of zygotic Cdk2 function could be explained by maternally derived Cdk2. The presence of a maternal Cdk2 contribution in the Drosophila egg has been demonstrated. To demonstrate the functional role of this maternal contribution, the development was analyzed of eggs derived from Cdk2 mutant females that had been rescued by periodic Hs-Cdk2 expression. These mutant females (Cdk22, Hs-Cdk2/Cdk23 or Cdk22, Hs-Cdk2/Cdk21) readily lay eggs as long as they are subjected to periodic heat shocks. However, after termination of periodic heat shocks, egg deposition decreases rapidly and stops completely within 2-3 days. This arrest of egg deposition is readily reversed within 7 days after resumption of periodic heat shocks (Lane, 2000).

The eggs from mutant females collected 1 day after the termination of periodic Hs-Cdk2 expression were fixed and stained for DNA. For comparison, the eggs from mutant females that had been maintained with periodic Hs-Cdk2 expression were also analyzed. In addition, eggs from w control females exposed to periodic heat shocks or 1 day after termination of these heat shocks were analyzed. The great majority of the eggs from these w control females reveal normal DNA staining patterns. Conversely, the majority of the eggs collected from mutant females (Cdk22, Hs-Cdk2/Cdk23 or Cdk22, Hs-Cdk2/Cdk21) display abnormal DNA staining patterns. The spatial distribution of nuclei and the appearance of chromatin is often aberrant, indicating that progression through the syncytial division cycles is severely perturbed in these embryos. This finding suggests that the maternal contribution is required during the syncytial division cycles. In addition, a significant fraction of embryos contains very few nuclei, suggesting that these embryos had failed to commence progression through the syncytial divisions (although it is not excluded that a minor fraction of these eggs was fixed while progressing normally through the first three cycles). Double labeling with an antibody recognizing a sperm tail epitope indicates that about two-thirds of these eggs with less than five nuclei are not fertilized. Compared to continuously heat-pulsed Cdk2 mutant females, those withdrawn from heat-shock treatment generate a higher fraction of eggs containing less than five nuclei at the expense of the eggs with normal appearance. Termination of periodic Hs-Cdk2 expression in Cdk2 mutant females, therefore, is accompanied by a transient production of eggs that cannot be fertilized followed by a rapid arrest of egg laying. A significant fraction (60%) of abnormal eggs is produced even when periodic Hs-Cdk2 expression is maintained. The production of abnormal eggs despite periodic Hs-Cdk2 expression is likely to reflect the fact that the germ line is refractory to induction of heat-shock genes during stages 10-12 of oogenesis. These observations demonstrate that Cdk2 expression is crucial for oogenesis and early embryogenesis (Lane, 2000).

The largest complementation group identified in this screen has not previously been implicated in Cyclin E function. Genetic and molecular analysis of this complementation group corresponds to the spen gene. Independent work has proven this suggestion (Wiellette, 1999). spen encodes a 600-kD ubiquituously expressed nuclear protein containing three RNP-type RNA binding domains and a novel characteristic C-terminal domain defining a family of homologous metazoan genes. Mutations in spen result in peripheral nervous system defects and interact with raf kinase signaling and E2F/DP transcription factors and with Cyclin E. It is attractive to speculate that spen is particularly important for the transition from cell proliferation to terminal differentiation. spen mutant ommatidia in the eye display the same defects as those resulting from the Sev-CycE transgene. The affected ommatidia are of variable composition, often lacking either R7 or one or more other photoreceptors, but also occasionally containing extra photoreceptors. Sev-CycE transgene expression forces differentiating cells through an extra cell cycle, presumably explaining the presence of extra cells. In addition, extra divisions of differentiating cells are likely to disturb the regular arrangement of the ommatidial cluster and consequently might cause apoptosis, potentially explaining the observed loss of cells as well. Similarly, coexpression of GMR-E2F1 and GMR-DP transgenes in all eye imaginal disc cells posterior to the morphogenetic furrow has been shown to result in ectopic BrdU incorporation and apoptosis. spen mutations dominantly enhance both the Sev-CycE and GMR-E2F1/DP rough eye phenotype. Conversely, spen mutations suppress the eye phenotypes resulting from GMR-dap expression in a CycE heterozygous background. While spen function opposes the mitogenic activity of CycE and dE2F1, it remains to be analyzed whether the phenotypic interactions observed between spen and Hox and Raf involve deregulated cell proliferation as well (Lane, 2000 and references therein).

The identification of mutations in Drosophila dE2F1 in this screen was expected on the basis of the large body of evidence demonstrating the tight functional relationship between Cyclin E and E2F/DP transcription factors. However, the fact that dE2F1 mutations result in enhancement rather than suppression of the Sev-CycE phenotype would not necessarily have been predicted since the results of genetic analysis in Drosophila so far have suggested that E2F/DP activity has a positive role in stimulating the transcription of S phase genes (Cyclin E, RNR2, DNA polalpha, PCNA, and Orc1) and cell proliferation. In contrast, the enhancement of the Sev-CycE phenotype observed with dE2F1 alleles points to a growth-suppressive role of dE2F1. Similarly, the E2F1 knock-out phenotype observed in mice has clearly demonstrated a tumor-suppressing function. Moreover, while vertebrate E2F/DP functions as a transcriptional activator in some promoters, it acts as a corepressor in conjunction with pRB in many other promoters. A decrease in E2F/DP levels, therefore, might also result in derepression of unknown proliferation-stimulating genes and synergy with ectopic Cyclin E expression (Lane, 2000 and references therein).

A cyclin-dependent kinase inhibitor, Dacapo, is necessary for timely exit from the cell cycle during Drosophila embryogenesis

In a screen for genes that interact with the Rap1 GTPase, a Drosophila gene, dacapo (dap) was identified that is a member of the p21/p27 family of cdk inhibitors. Unlike mammalian cdk inhibitors studied to date, dap is essential for normal embryonic development. Dacapo inhibits cyclin-cdk activity in vitro. Overexpressing dap during eye development interferes with cell cycle progression and interacts genetically with the retinoblastoma homolog (Rbf) and cyclin E. dap expression in embryos parallels the exit of cells from the cell cycle. dap mutant embryos delay the normal cell cycle exit during development; many cells complete an additional cycle and subsequently become quiescent. Thus, dap functions during embryogenesis to achieve a precisely timed exit from the cell cycle (de Nooij, 1996).

Most cell types in multicellular eukaryotes exit from the mitotic cell cycle before terminal differentiation. This study shows that the dacapo gene is required to arrest the epidermal cell proliferation at the correct developmental stage during Drosophila embryogenesis. dacapo encodes an inhibitor of cyclin E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors. dacapo is transiently expressed beginning late in the G2 phase preceding the terminal division (mitosis 16). Mutants unable to express the inhibitor fail to arrest cell proliferation after mitosis 16 and progress through an extra division cycle. Conversely, premature dacapo expression in transgenic embryos results in a precocious G1 arrest (Lane, 1996).

Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation

Most cells of the dorsal epidermis exit from the mitotic cycle after division 16 in Drosophila embryogenesis. This exit is dependent on the down-regulation of Drosophila cyclin E (cycE) during the final mitotic cycle. Ectopic expression of cycE after the final mitosis induces entry into S phase and reaccumulation of G2 cyclins and results in progression through a complete additional cell cycle. Conversely, analyses in cycE mutant embryos indicate that cyclin E is required for progression through S phase of the mitotic cycle. Moreover, endoreplication, which occurs in late wild-type embryos in the same pattern as cycE expression, is not observed in the mutant embryos. Therefore, Drosophila Cyclin E, which forms a complex with the Cdc2 kinase, controls progression through S phase and its down-regulation limits embryonic proliferation (Knoblich, 1994).